TECHNICAL TECHNICAL
REPORTS: WASTE
MANAGEMENT
REPORTS
Humic Acid Formation in Artificial Soils Amended with Compost at Different Stages of
Organic Matter Evolution
Fabrizio Adani* and M. Spagnol DiProVe–Università di Milano
A composting process was conducted under optimal
conditions for 150 d, obtaining three biomasses at different
levels of maturity: raw material (RM), fresh compost obtained
after 11 d of composting (FC), and evolved compost (EC)
obtained after 150 d of composting. During the composting
process, HAs were extracted and fully characterized by mass
balance, DRIFT, and 1H and 13C-nuclear magnetic resonance
spectroscopy. Each compost sample was incubated for 180 d
in an artificial soil, after which HA extraction was repeated
and characterized. To compare composts containing different
amounts of labile organic matter (OM), an equal amount
of unhydrolyzable OM was added to the soils. Our results
indicated that compost HAs consist of a biologically and
chemically stable fraction (i.e., the unhydrolyzable HA [U-HA])
and a labile fraction, whose relative contents depended on the
composting duration. Humic acid from more EC contained a
higher amount of recalcitrant fraction (aromatic carbon) and
a lesser amount of labile fraction (aliphatic carbon) than HA
from RM and FC. These results suggest that the humification
process during composting preserves the more recalcitrant
fraction of the compost-alkali soluble/acid insoluble fraction
(HA-fraction). Incubation of composts in soil showed that due
to the higher labile fraction content, HAs from raw material
were more degraded than those from EC. The abundance of
labile carbon of soil amended with less-evolved compost (RM
and FC) allowed the more recalcitrant fractions of U-HA to
be more preserved than in EC. These results suggest that lessevolved compost could contribute more than well evolved
compost to the stable soil OM.
Copyright © 2008 by the American Society of Agronomy, Crop Science
Society of America, and Soil Science Society of America. All rights
reserved. No part of this periodical may be reproduced or transmitted
in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system,
without permission in writing from the publisher.
Published in J. Environ. Qual. 37:1608–1616 (2008).
doi:10.2134/jeq2007.0108
Received 28 Feb. 2007.
*Corresponding author ([email protected]).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
S
oil quality depends on the soil organic matter (SOM)
quantity, quality, and dynamics (Lal, 2000). Up to 70 to
80% of SOM in mineral soil is composed of humic substances
(HS) (Piccolo, 2002). Compost can be beneficial for soil
because it improves soil properties and contributes to the soil
humus balance (Giusquiani et al., 1995; Quèdraogo et al.,
2001; Leifeld et al., 2002). The definition of HS is only used
for operational purposes because it is based on the solubility in
the aqueous solutions used as extractants (Piccolo, 2002), and
the term humic acid (HA) is used to indicate the HS soluble
in dilute alkali but insoluble in dilute acid (pH < 1) (Piccolo,
2002). Humic acid is a negatively charged colloid, is recalcitrant
to biodegradation, and can be stored in soil for long periods
(Qualls, 2004). Because of these properties, HA plays an
important role in determining soil characteristics by influencing
its chemical, physical, and biological properties. Consequently,
compost HA is considered an indicator of the ability of compost
to contribute to SOM balance (Adani et al., 2006a).
Humification during composting has been the subject of
extensive study (Sugahara and Inoko, 1981; Inbar et al., 1990;
Chen and Inbar, 1993; Chefetz et al., 1999; Genevini et al.,
2002a). However, the nature of the humification process during
composting remains unclear. Furthermore, the end products of
compost humification differ from those of soils (González-Vila
et al., 1999; Almendros et al., 2000).
It was previously reported that HA is formed ex novo during composting (Chen and Inbar, 1993). This report was based
on the findings that the relative content of the HA fraction increased during composting. This interpretation of the humification process refers to the classical route of HA formation, which
hypothesizes that bio-macromolecules are broken up into small
constituents and then recombined to form chemically complex
“geopolymers” (Senesi and Loffredo, 1999). However, when
considering the absolute content of HA, there is evidence that
during composting this content does not increase notably (Adani
et al., 1999; González-Vila et al., 1999).
More recently, it was shown that humification during the
composting process should be interpreted as the concentrating
DiProVe–Università di Milano, via Celoria 2, 20133 Milano, Italy.
Abbreviations: DRI, dynamic respiration index; EC, evolved compost; FAC, fulvic
acid carbon; FC, fresh compost; HA, humic acid; HAC, humic acid carbon; HS, humic
substance; HUC, humic carbon; NHC, nonhumified carbon; OM, organic matter; RM,
raw material; SEC, soil amended with evolved compost; SFC, soil amended with
fresh compost; TEC, total extractable carbon; TOC, total organic carbon; U-TOC,
unhydrolyzable total organic carbon.
1608
of pre-existing nonhydrolyzable HA molecules (proto-HA)
(Chefetz et al., 1999; Genevini et al., 2002a, b). Also, during
composting, modifications of nonhydrolyzable HA, such as
decarboxylation, demethoxylation, and a general removal of
O-alkyl and alkyl carbons, occur (González-Vila et al., 1999;
Chefetz et al., 1999; Genevini et al., 2002a).
This view of the humification process does not conflict with
more recent studies on the nature of humic substances that
indicate that HA is composed of low-molecular-weight hydrophobic and amphiphilic compounds (lignin, polysaccharides,
lipids, and peptides) derived from the degradation and decomposition of dead biological material, arranged in supramolecular
structures (Piccolo, 2002; Simpson, 2002; Sutton and Sposito,
2005). Therefore, based on our previous work (Genevini et al.,
2002a, 2002b; Chefetz et al., 1999; Adani et al., 2006b) and
taking into consideration the new interpretation of the HA
structure, it can be concluded that compost HAs are represented by a mix of low-molecular-weight molecules structured in
a supramolecular association and whose composition depends
on the duration of the degradation processes (the composting
process removes more labile fraction).
The production of fresh compost by high-rate composting—compost produced without the curing phase—represents a new trend in composting (Scaglia et al., 2000). This
composting strategy was devised to reduce composting time
and produce a hygienic, stable, and safe product.
It can be argued that if humification of OM takes place during the curing phase (Chen and Inbar, 1993), then the humus
content of fresh compost is limited compared with mature
compost. However, if humification is considered as the conservation and modification of more recalcitrant biomolecules, then
the differences in the contribution to the soil–humus balance
depend only on the relative concentration of these molecules.
The aim of this work was to study the effects of the composting process on the concentration and preservation of
pre-existing HA (nonhydrolyzable HA) in compost. This
study investigated the effects of compost, obtained at different
degrees of evolution (fresh compost [FC] obtained after 11 d
of composting and evolved compost [EC] obtained after 150
d of composting), on the quantity and quality of soil HA.
Materials and Methods
Compost Preparation
The raw material (RM) used was an organic mix consisting
of municipal solid waste and green waste collected from the city
of Milano (North Italy) mixed together in a ratio of 2.5:1 (wet
weight) or 1:1 (dry weight). Raw material (RM) was composted in a laboratory adiabatic reactor (Adani et al., 2004). Fresh
compost was obtained at the end of the high-rate composting phase (11 composting days) when compost attained the
medium biological stability as required by the regional rules
of Lombardia (North Italy) (Scaglia et al., 2000). This product was characterized by an oxygen uptake rate expressed as a
dynamic respiration index (DRI) of 1000 mg O2 kg−1 organic
matter (OM)−1 h−1 (Adani et al., 2004). More EC was obtained
after a total of 150 composting days (high-rate composting plus
a curing phase). This product exhibited a DRI of 200 mg O2 kg
OM−1 h−1, which was lower than the DRI that defines mature
compost (500 mg O2 kg OM−1 h−1) (Adani et al., 2004).
All matrices (RM, FC, and EC) were sampled during composting, following procedures described by The U.S. Composting Council (1997). Samples were vacuum dried at 60°C,
ground to a Ø <0.5 mm, and stored for subsequent use.
Incubation Test
The soil used for the incubation tests was a sandy mineral
substrate consisting of sand (particle size ∅ = 0.5–0.8 mm, pH
7) and clay (bentonite-montmorillonite–like mineral sieved at
Ø < 1 mm, pH 7, CEC = 6.5 cmol+ kg−1) (Adani and Ricca,
2004). Sand and clay were mixed in a ratio of 9:1 (w/w). An
artificial medium was chosen to avoid contamination from
SOM (Almendros and Dorado, 1999) and does not represent a
natural soil (Adani and Ricca, 2004) and needs future comparison with natural soil. Organic amendments were added to the
soil by normalizing each biomass to the nonhydrolyzable fraction of the total organic carbon (TOC) (see below for analytical
details) and named the total unhydrolyzable carbon (U-TOC).
An equal amount of this fraction (3 g kg−1 dm−1) was added
to each sample amended with different composts. This choice
was based on previous research (Adani and Ricca, 2004) indicating that the U-TOC was the stable OM fraction remaining
after a medium-term degradation process (6 mo). Moreover,
the nonhydrolyzable OM fraction (the biochemically protected
OM) was shown to be the recalcitrant fraction that contributes,
together with physically and chemically stabilized OM, to the
stable soil OM (Six et al., 2002; Mikutta et al., 2006). Therefore, the ability of different organic matrices characterized by
different carbon contents available to contribute to the stable
OM of soil could be compared using this method.
Using 5-L pots, incubation tests were replicated twice for each
sample. Each pot was filled with approximately 2.5 kg of soil (dry
matter). Soils were wetted with water and kept at 60% (w/w) of
the maximum water-holding capacity. Water content was corrected gravimetrically every 3 d. Pots were incubated for 180 d
in a chamber at 26 ± 2°C under dark conditions. During the
incubation tests, soils amended with composts at different levels
of maturity—SRM (soil amended with RM), soil amended with
fresh compost (SFC), and soil amended with evolved compost
(SEC)—were sampled at time 0 (T0), 60 (T2), 90 (T3), 120
(T4), and 180 d (T6), respectively. Each sample weighed approximately 100 g, except for the last sample, which weighed 150
g and was formed by three subsamples taken from each replicate.
Soils were vacuum-dried at 65°C and then used for analytical
determination. Analyses were performed in triplicate.
Organic Matter and Organic Carbon Fractionation
The OM was fractionated into its macromolecules using the
wood analysis method adapted for soil (Adani and Ricca, 2004).
Three consecutive extractions were performed on soil samples.
The fraction soluble in benzene/ethanol mixture (2:1 v/v) and
16.87 mol L−1 ethanol (soluble lipids, waxes, soluble tannins,
Adani & Spagnol: Humic Acid Formation in Artificial Soils Amended with Compost
1609
proteins, and part of fulvic acids) was solubilized by the first extraction step (fraction I); protein, hemicelluloses, and part of fulvic
acids (fraction II) were solubilized in hot 0.94 mol L−1 H2SO4
under reflux for 2 h (second step); and cellulose (fraction III) was
solubilized in 13.50 mol L−1 H2SO4 at 4°C for 24 h (third step).
The lingo-humic complex (fraction IV), insoluble in any solvent,
was left in the residual fraction (lingo-humic complex plus acidinsoluble ashes) and quantified by ignition at 650°C of an aliquot
of this fraction.
Isolation of the ligno-humic complex was performed for
RM, composts, and soils. Volatile solids and ash content for
the three organic matrices were determined by ignition loss at
650°C (APHA, 1992).
Humic acid (HA) and unhydrolyzable humic acid (U-HA)
extractions were performed as described previously. Humic acid
was directly extracted from RM, compost, and soil samples,
whereas UHA was extracted for RM, compost, and soil samples
from the ligno-humic complex obtained as described previously.
In brief, 2 g of dried compost (10 g for soils) was extracted in
a 250-mL Erlenmeyer flask with 100 mL of 0.1 mol L−1 NaOH
and 0.1 mol L−1 Na4P2O7 solution at 65°C for 24 h under N2 in
a Dubnoff thermostatic bath at 100 oscillations min−1 (Adani and
Ricca, 2004). The sample was cooled to room temperature in a
150-mL centrifuge tube and centrifuged at 13,000 rpm for 20
min. The extraction was repeated by adding distillate water until
the supernatant was clear. The supernatant solution (alkaline
extract) (TE or U-TE) was filtered through a 0.45-μm Millipore
filter. The insoluble humic fraction (HU or U-HU) was retained
and washed until a neutral pH was obtained and then stored. The
alkaline extracts were then acidified to below pH 1.5 and centrifuged at 5000 rpm for 20 min. The insoluble fractions (HA or
U-HA) were washed with distilled water until a neutral pH was
obtained and followed by an extraction with 2.82 mol L−1 HF
for 24 h at room temperature to remove inorganic constituents
(silicates). Then, HA (or U-HA) was washed to remove excess of
HF with distilled water until a neutral pH was obtained, vacuum
dried at 60°C, and weighed. The ash content of all extracted fractions was less than 1%. The residual alkali-soluble/acid-soluble
fraction was purified by chromatography using a polyvinylpirrolidone column, obtaining two fractions: fulvic acid (FA) and
nonhumified material (NH).
The alkali-soluble/acid-soluble fraction obtained starting from
the lingo-humic fraction represented the U-NH, as purification by
chromatography using a polyvinylpirrolidone was omitted because
FA is solubilized during OM fractionation (see Fraction I and II).
All fractions obtained during the HA and U-HA extractions were quantified by organic carbon determinations (Ciavatta et al., 1990) obtaining total organic carbon (TOC), total
extractable carbon (TEC), humic acid carbon (HAC), fulvic
acid carbon (FAC), humin carbon (HUC), and corresponding
unhydrolyzable-C. Nonhumified carbon (NHC) was calculated as [TEC – (FA + HA)] and U-NHC as (U-TEC – U-HA).
Variations in the content of each single fraction during
composting and soil incubation were calculated from the start
and end absolute fraction contents (g) and reported as the
relative increase or decrease (g kg−1).
1610
Diffuse Reflectance Infrared Fourier Transformed,
1
H–Nuclear Magnetic Resonance, and 13C–Nuclear
Magnetic Resonance Spectroscopy
Diffuse reflectance infrared fourier transformed (DRIFT)
spectra were recorded with a FT/IR-300 E JASCO spectrometer
equipped with a Diffuse Reflectance attachment (Pike Technologies, Inc, Madison, WI). The samples, a 7-mg sample and 700
mg potassium bromide (FT grade; Aldrich Chemical Co, St.
Louis, MO), were finely ground using a Wing-L-Bug agate ball
mill for 10 min (Specamill-Greseby-Specac, Kent, UK). The
spectra were acquired in the 4000 to 600 cm−1 range with 4 cm−1
resolution, and 100 scans were performed on each acquisition. A
background spectrum of finely powdered potassium bromide was
recorded using the same instrument settings. Peak assignments
were made according to Adani and Ricca (2004). In brief, the
bands at 2920 to 2850 cm−1 were attributed to aliphatic chains
and peaks at 1710 cm−1 to the stretching of C=O of ester, ketons,
and acid carboxylic. Bands at 1666 cm−1 and 1542 cm−1 were attributed to the stretching of C=O of amide (amide I) and to deformation of N–H bond and stretching of C=N in amide (amide
II).The bands at 1600 cm−1 and 1420 cm−1 were due to the C=C
stretching of aromatic rings. The peak at 1516 cm−1 was typical of
an aromatic structure (e.g., lignin) (C=C stretching). The peak at
1456 cm−1 was attributed to C–H bond deformation in aliphatic
chains. The bands at 1273 cm−1 and 1215 cm−1 were due to C–O
bonds of phenol. The band at 1032 cm−1 was attributed to C–O
stretching in the polysaccharide, and the band at 840 cm−1 was
due to C–H deformation out of the plane (lignin structures).
1
H-NMR spectra were recorded using a Bruker AC 300
spectrometer at 300 MHz under homogated decoupling
conditions, the HOD peak produced by water impurities
and proton exchange being irradiated. The radio frequency
level of HOD irradiation was optimized for each sample. The
NMR samples were prepared by dissolving 10 mg of sample
in 0.5 mL solutions of NaOD 0.5 mol L−1. 1H-NMR gave
semi-quantitative data, using calculations of the typical area
corresponding to three regions. These regions were 0.5 to 3.3
ppm (alkyl protons, protons attached to carbon by an α bond
to an aromatic ring or carboxylic groups, Hα), 4.8 to 3.3 ppm
(protons attached to carbon bearing oxygen or nitrogen), and
7.8 to 6.5 ppm (aromatic and olefinic protons, HAr).
13
C NMR spectra were recorded using a Bruker AC 300
spectrometer at 75.43 MHz. The NMR samples were prepared
by dissolving approximately 50 mg of HA-like or unhydrolyzedalkali-soluble HA-like in 0.5 mL of NaOD 1.5 mol L−1. A quantitative intensity distribution was achieved by using the inversegated decoupling method: 0.23 s acquisition time, 45° pulse, 2 s
relaxation delay, line broadening at 20 Hz, and a total acquisition
time of 48 h. All chemical shifts were quoted with reference to
internal 4,4-dimetyl-4-silapentanesulfonic acid (Na-salt).
For a semi-quantitative approach, the 13C-NMR spectra
were subdivided into six regions. The signals ranging between
190 and 165 ppm were attributed to carboxyl and amide carbons. Peaks between 165 and 145 ppm arose from C=C–O of
phenols. Peaks between 145 and 110 ppm were due to aromatic
Journal of Environmental Quality • Volume 37 • July–August 2008
carbons (C=C). The signals ranging between 110 and 60 ppm
were due to C–O of carbohydrates and aliphatic alcohols.
Peaks between 60 and 50 ppm arose from methoxyl carbon
(O–CH3). The signals ranging between 50 and 0 ppm were due
to alkyl carbons. Variations in H and C contents were calculated as [(starting content – final content)/starting content] × 100.
Results
Composting Process
The decrease of volatile solids during composting indicated
that degradation occurred during the whole composting proFig. 1. Dry matter fractionation for raw material (RM), fresh compost (FC),
cess (Fig. 1). The composting process proceeded through the
and evolved compost (EC). I: fraction soluble in organic solvent; II:
decomposition of the readily degradable organic fraction (celfraction soluble in 0.94 mol L−1 H2SO4; III: fraction soluble in 13.50
lulose, hemicellulose, proteins, and lipids; i.e., fraction I, II and
H2SO4; IV: fraction insoluble in any solvent. VS: volatile solids.
III) (Almendros et al., 2000), whereas the lingo-humic complex
Both 1H-NMR and 13C-NMR (Tables 2 and 3) confirmed
(fraction IV) was concentrated, and, as expected, the ash content
what was previously known about the chemical modification of
remained constant (Fig. 1). Composting was also found to afHA and U-HA during composting. The composting process of
fect the C fractions because these all decreased during compostHA of RM compared with HA of EC found a decrease of the aliing (Table 1). In contrast, U-TOC was relatively stable during
phatic proton content (−21.7%) and an increase of the O/N-Ccomposting, decreasing by only 136 g kg−1 (Table 1). Because
H (+108%) and aromatic protons (+160%) (Table 2). This trend
U-TOC represented the sum of all U-C fractions, the large
was confirmed by 13C-NMR, which revealed a relative decrease
decrease of the alkali-soluble fraction (U-TEC = U-HAC + Uof alkyl-C content (−62.2%) and an increase of carboxyl, phenol,
NHC) registered during composting could only be explained
aromatic, O-methoxyl, and O-alkyl carbons (+20.9, +6.8, +11.5,
by an increase of the alkali-insoluble fraction (i.e., U-humin C)
+45.8, and +11.4%, respectively) in the HA fraction (comparing
(Table 1). These data contrasted with our previous findings that
HA of RM vs. HA of EC) (Table 3) (Genevini et al., 2002a).
indicated that during the composting process, soluble U-HA
The chemical pretreatment of the raw material before HA extracis formed from the insoluble U-HU (Genevini et al., 2003).
tion had similar effects on the composting process (Chefetz et al.,
A similar observation supporting our most recent findings has
1999; Genevini et al., 2002a, 2002b). Our results demonstrate
previously been reported (González-Vila et al., 1999). As demthat the U-HA from RM was similar to HA extracted from EC,
onstrated by our DRIFT spectra data (Fig. 2), composting also
except for O-alkyl-C and alkyl-C, as shown by 1HNMR and
qualitatively modifies HA. When compared with HA extracted
13
C-NMR (Tables 2 and 3). These differences could be due to the
from FC and raw material (RM), the HA DRIFT spectra of EC
presence of acid-hydrolyzable O/N–alkyl-C and alkyl-C fractions
indicated a decrease of aliphatic chains content and an increase
in HA at the end of composting (EC-HA), which may be of miof N-containing and lignin-derivates molecules (González-Vila
crobial origin (González-Vila et al., 1999).
et al., 1999). The increase of N-molecules was most likely due to
The biological process and the chemical treatments suggested
molecules of a microbial origin (González-Vila et al., 1999).
that the origin of HA in compost throughout the biological
Purification of the parent material before HA extraction redegradation of the labile part of HA is due to the preservation
moved the HA labile fractions and simulated the biological proof pre-existing recalcitrant fractions (e.g., U-HA) (Chefetz et
cess. This confirmed our previous findings (Chefetz et al., 1999;
Genevini et al. 2002a, 2002b; Adani et
Table 1. Absolute organic carbon and unhydrolyzable organic carbon contents for the different
al., 2006b) (i.e., a reduction of aliphatic
carbon fractions during the composting process.
chains and N-containing molecules and
Compost
TOC†
TEC
HAC
FAC
NHC
HUC
an increase of lignin-derived molecules
g
as revealed by the comparison of DRIFT
RM‡
600.9 ± 11.5c§ 301.7 ± 3.7c 107.6 ± 1.9c 9.2 ± 0.2b 184.9 ± 4.2c
299.3 ± 12.1b
spectra of HA and corresponding U-HA) FC
411.4 ± 17.4b 133.9 ± 4.3b
56.4 ± 1.9b 2.9 ± 0.2a 74.5 ± 4.8b
277.5 ± 17.9b
(Fig. 2). The decrease of N-molecules
EC
227.8 ± 16.43a 77.0 ± 3.7a
33.6 ± 0.1a 2.3 ± 0.5a 41.1 ± 3.8a
150.8 ± 16.8a
seems to confirm their microbial origin
U-TOC¶
U-TEC
U-HAC
U-FAC
U-NHC
U-HUC
in HA because this fraction could only be RM
126.2 ± 6.2b
74.3 ± 7.4c
48.6 ± 1.9c
nd
25.7 ± 7.6b
51.9 ± 9.62a
removed by a chemical treatment. Also, it FC
123.3 ± 2.8b
61.05 ± 4.4b
38.3 ± 1.9b
nd
22.7 ± 4.8b
62.3 ± 5.2b
109.0 ± 3.7a
36.03 ± 0.0a
26.7 ± 1.01a
nd
9.4 ± 1.0a
72.9 ± 3.7c
could not be removed during the biologi- EC
† FAC, fulvic acid carbon; HAC, humic acid carbon; HUC, humic carbon; NHC, nonhumified carbon; TEC,
cal process due to the presence of OMdegrading microorganisms that continu- total extractable carbon; TOC, total organic carbon.
ally contributed through their biomass to ‡ EC, evolved compost; FC, fresh compost; RM, raw material.
§ Means followed in the same column by the same letter are not statistically different (p < 0.05)
enrich HA with N-containing molecules according to Tukey test.
(González-Vila et al., 1999).
¶ Prefix U indicates unhydrolyzable fraction.
Adani & Spagnol: Humic Acid Formation in Artificial Soils Amended with Compost
1611
al., 1999). Unhydrolyzable HA
extracted from the final stage of
composting (U-HA from EC) was
more aromatic and less aliphatic
than U-HA from RM and FC
(Table 3), indicating that U-HA
was modified during composting.
From 13C-NMR it can be seen that
this modification was due to the
loss of the major part of the alkyl-C
(a decrease of 56%), passing from
RM to EC. This was confirmed
by calculating the absolute content
of the different C-types by using
data reported in Table 3 and Table
1. From this calculation it could
be shown that alkyl-C accounted
for more than 50% of the total C
loss, followed by an aryl-C loss of
16.7%, a carboxyl-C loss of 16.3%,
an O-methoxyl-C loss of 10.2%,
and a N/O-alkyl-C loss of 3.4%.
Phenol-C remained unchanged.
Consequently, the relative concentrations of phenol and aryl-C
increased by 79.7% and 34.7%,
respectively. This trend was further confirmed by the good correFig. 2. DRIFT spectra for humic acids (left) and unhydrolyzable-humic acid (right) extracted from raw
lation found between aryl-C and
material (RM), fresh compost (FC), and evolved compost (EC).
phenol-C vs. alkyl-C (alkyl-C vs.
Table 2. Integrated area (%) of different hydrogen types of humic
aryl-C: r = −0.99, P < 0.01; alkylacid (HA) and unhydrolyzable humic acid (U-HA) extracted from
C vs. phenol-C: r = −0.97, P < 0.01). These results suggest that
compost at different stages, and incubated soil with composts at
the start and end of the trials (1H–nuclear magnetic resonance).
a fraction formed mainly by the alkyl-C of the U-HA becomes
H–C–O
more hydrolyzable as the biological process (composting) proAryl-H
H–C–N
Alkyl-H
ceeds. The loss of this fraction from the U-HAC could also
Sample
(7.4–6.0 ppm) (4.8–3.0 ppm) (3.0–0.5 ppm)
explain the decrease of U-HAC content in EC as previously
Composts
shown by mass balance (Table 1).
HA-RM†
5.8
8.6
85.6
HA-FC
4.8
4.6
90.5
HA-EC
15.1
17.9
67.0
U-HA-FC‡
18.0
23.2
58.7
U-HA-RM
14.8
23.5
61.7
U-HA-EC
28.8
32.8
38.3
Soils
HA-SRM§ T0¶
5.8
8.6
85.6
HA-SRM T6
9.4
11.1
79.5
HA-SFC T0
4.8
4.6
90.5
HA-SFC T6
13.1
22.0
64.8
HA-SEC T0
15.1
17.9
67.0
HA-SEC T6
14.9
25.5
59.6
U-HA-SRM T0
14.8
23.5
61.7
U-HA-SRM T6
19.9
25.6
54.5
U-HA-SFC T0
18.0
23.3
58.7
U HA-SFC T6
18.4
29.7
51.8
U-HA-SEC T0
28.8
32.9
38.3
U-HA-SEC T6
23.1
27.2
49.6
† RM, raw material; FC, fresh compost; EC, evolved compost.
‡ Prefix U indicates unhydrolyzable fraction.
§ Soil incubated with: raw material (SRM), fresh compost (SFC), and evolved
compost (SEC).
¶ T0, T6: soil sampled after 0 and 6 mo of incubation.
1612
Soil Incubation
As a consequence of microbial activity, soil incubated with raw
material (SRM) exhibited a TOC decrease after 6 mo of incubation (Fig. 3). A general decrease of all C fractions composing the
TOC occurred during the incubation (Fig. 3). In particular, TEC,
HAC, FAC, NHC, and HUC decreased by 622, 707, 807, 577,
and 497 g kg−1, respectively. In contrast, recalcitrant carbon (UTOC) remained relatively constant during the 6-mo incubation
period (Table 4). This is important because it justifies the approach
used in this experiment whereby we added an equal amount of
U-TOC (the stable fraction of carbon over a medium-term incubation period), independent of the degree of compost evolution
(Adani and Ricca, 2004), to each sample. We noted deceases in
U-TEC, U-HAC, and U-NHC of 581, 607, and 539 g kg−1, respectively, whereas U-HUC increased by 745 g kg−1.
Soil amended with fresh compost exhibited a similar trend
to SRM (Fig. 3) in that all carbon fractions decreased during
incubation, but less than was the case in the other samples.
Journal of Environmental Quality • Volume 37 • July–August 2008
Table 3. Integrated area (%) of different carbon types of humic acid (HA) and unhydrolyzable humic acid (U-HA) extracted from compost at
different stages and incubated soil with composts at the start and end of the trials (13C–nuclear magnetic resonance).
Carboxyl-C
Phenol-C
Aryl-C
O-alkyl-C
Sample
(190–165 ppm) (165–145 ppm) (145–110 ppm) (110–60 ppm)
Composts
HA-RM†
9.8
3.3
16.5
5.9
HA-FC
11.9
3.9
14.2
12.2
HA-EC
12.4
10.1
28.0
17.3
U-HA-RM‡
14.6
8.9
29.4
5.1
U-HA-FC
10.9
9.6
32.3
10.4
U-HA-EC
13.0
16.0
39.6
6.4
Soils
HA-SRM§ T0¶
9.8
3.3
16.5
5.9
HA-SRM T6
20.4
3.3
23.6
20.0
HA-SFC T0
11.9
3.9
14.2
12.2
HA-SFC T6
12.8
5.7
19.9
19.3
HA-SEC T0
12.4
10.1
28.0
17.3
HA-SEC T6
18.8
11.4
22.3
23.7
U-HA-SRM T0
14.6
8.9
29.4
5.1
U-HA-SRM T6
14.0
13.3
32.6
8.8
U-HA-SFC T0
10.9
9.6
32.3
10.4
U-HA-SFC T6
10.9
13.6
36.8
5.1
U-HA-SCE T0
13.0
16.0
39.6
6.4
U-HA-SCE T6
13.9
16.8
41.1
2.3
† EC, evolved compost; FC, fresh compost; RM, raw material.
‡ Prefix U indicates unhydrolyzable fraction.
§ Soil incubated with raw material (SRM), fresh compost (SFC), and evolved compost (SEC).
¶ T0, T6: soil sampled after 0 and 6 mo of incubation.
Total organic carbon decreased by 484 g kg−1, whereas TEC,
HAC, FAC, NHC, and HUC decreased by 689, 577, 608,
373, and 358 g kg−1, respectively. Unhydrolyzable TOC remained constant during the 6-mo incubation period (Table
4). In contrast, U-TEC, U- HAC, and U-NHC decreased by
611, 590, and 645 g kg−1, respectively, whereas U-HUC increased by 471 g kg−1 (Table 4).
Soil amended with EC showed a different trend with respect
to SRM and SFC. Carbon fractions decreased, but this decrease
was less pronounced than observed in SFC and SRM. Total
organic carbon, TEC, HAC, and HUC decreased by 276, 55,
560, and 335 g kg−1, respectively (Fig. 3), whereas NHC increased by 302 g kg−1 (Fig. 3). Unlike the other two samples, UTOC decreased significantly (−211 g kg−1). Similarly, U-TEC
and U-HAC decreased significantly by 351 and 500 g kg−1,
respectively, whereas U-NHC increased (428 g kg−1) (Table 4).
After 6 mo of incubation of the amendments in artificial soils
(sample T6), HA composition exhibited a modified DRIFT
spectrum (Fig. 4). This indicated that incubation removed the labile fraction (i.e., lipids from HA for SRM and SFC and concentrated N-containing and lignin-derived molecules). In contrast,
the DRIFT spectrum of HA extracted from the EC-amended
soils (SEC) after 6 mo of incubation did not show an appreciable
difference to that of HA extracted at time zero (T0).
These observations were confirmed by 1H-NMR and 13CNMR spectra of HA that changed significantly for SRM and SFC
after 6 mo of incubation. The alkyl fraction decreased, and the
O/N-alkyl and aryl/phenol fractions increased (Tables 2 and 3).
On the other hand, SEC did not exhibit much variation (Table 3).
As demonstrated by the composting process and the DRIFT
spectra (Fig. 4), chemical pretreatment of soils before HA ex-
O-methoxyl-C
(60–50 ppm)
Alkyl-C
(50–0 ppm)
7.2
7.3
10.5
10.6
11.7
10.9
57.2
50.5
21.6
31.3
25.1
13.8
7.2
8.0
7.3
11.6
10.5
7.0
10.6
12.8
11.7
14.1
10.9
12.2
57.2
24.6
50.5
30.7
21.6
16.7
31.3
18.4
25.1
19.3
13.8
13.6
traction had an effect similar to biological processes (Fig. 3).
The NMR data demonstrated that during composting of SRM
and SFC, the alkyl fraction became more hydrolyzable, and
phenol-C increased with prolonged incubation times (Table 3).
Discussion
Degradation processes during composting affect the HA content and its quality. The HA content decreases due to the degradation of labile organic molecules forming more recalcitrant (i.e., the
unhydrolyzable) HAs (Chefetz et al., 1999). Therefore, HAs were
formed by a labile fraction and a recalcitrant fraction that was preserved (Binachessi da Chuna-Santino and Bianchini Júnior, 2002).
We have found that HA from more evolved compost contains
more recalcitrant fraction (aromatic carbon) and less labile fraction
(aliphatic carbon) than HA from RM and FC, indicating that HA
composition is dependant on the composting duration.
Labile fractions consist of a free and a hydrolyzable-lipid fraction (free fatty acids and esterifies lipids) that account for approximately 67% (13C-NMR data) of total HA lost during composting
(Genevini et al., 2002a). O/N-alkyl C contributed modestly to
the composition of the labile fraction as its content increased during composting (see 13C-NMR data in the Results section). The
degradation of HA-carbohydrates during composting is well documented in the literature (González-Vila et al., 1999; Almendros
and Dorado, 1999). It is likely that the O/N-alkyl fraction of HA,
for example the carbohydrates and proteins that are hydrolyzable,
also contribute to the labile-HA fraction. However, this fact was
probably masked by the production of newly formed alkali-soluble
molecules of microbial origin that contributed to HA molecules
(González-Vila et al., 1999). In addition, the strong decrease of the
alkyl fraction determining a strong relative increase of the other
Adani & Spagnol: Humic Acid Formation in Artificial Soils Amended with Compost
1613
The unhydrolyzable recalcitrant fraction of HA, the U-HA, is
mainly composed of highly polymerized aromatic lignin-derived
molecules (acid-resistant lignin) (Leary et al., 1986) and carbohydrates (Kniker and Lüdemam, 1995), probably forming an unhydrolyzable cross-linked network (e.g., alkali soluble part of the cell
wall of plant material) (Adani and Ricca, 2004; Adani et al., 2006c;
Adani et al., 2007). The U-HA was not stable during the composting processes. Our results suggested that the alkyl fraction contributed above all to the loss of the U-HA. This fact contributed to the
relative increase of the aromatic-C content of the U-HA.
Composts in soils were degraded at a rate depending on their
degree of evolution (Pascual et al., 1999), as RM was degraded
to a greater extent than EC. The use of fresh material allowed for
higher TOC and, more significantly, higher U-TOC contents at
the end of the incubation. These data are not surprising because
TOC dosed with RM and FC was much higher than that dosed
with EC. On the other hand, the U-TOC results are unexpected
because it was dosed with the same amount for the different samples. This suggested that the biological stability of the biochemically protected carbon (the U-TOC) depended on the amount of
available-C for microorganisms. Therefore, the higher amounts
of labile-C from samples with RM and FC allowed for the preservation of the more stable-C.
Humic acids were degraded at different rates depending on
their content in the labile fractions (Almendros and Dorado,
1999). As composting removed the HA labile fraction, HA
from EC showed the lowest rate of degradation when incubated in soil. Nevertheless, because of the higher level of HA
dosed, soil amended with RM showed a higher HA content at
the end of the incubation period. However, the final content
Fig. 3. Absolute organic carbon and unhydrolyzable-carbon fraction
of HA from FC and EC was similar. Humic acid degradation
contents during incubation of composts in soils. Soil amended
of SFC proceeded mainly by degradation of the alkyl fraction,
with raw material (top); soil amended with fresh compost
(middle); soil amended with evolved compost (bottom). FAC,
whereas for SEC, the aromatic fraction was mainly degraded.
fulvic acid carbon; HAC, humic acid carbon; HUC, humic carbon;
When the U-HA was considered, the two samples showed
NHC, nonhumified carbon; TEC, total extractable carbon; TOC,
interesting
results. Although at the end of the incubation period
total organic carbon.
U-HA contents were the same, the joint decrease of U-HA and
fractions may have contributed to this apparent nondegradation
U-TOC registered for SEC suggested that U-HA was probably
of the O/N-alkyl fraction. This may have also occurred for the
degraded. In contrast, SFC showed constant U-TOC values,
aromatic fraction (aryl-C + Phenol-C) but is unlikely because the
suggesting that the U-HA fraction was not degraded but probincrease of this fraction was double that of O-alkyl-C, indicating a
ably was insolubilized.
degradation of recalcitrant fraction.
Humic acid from EC was expected to be more recalcitrant
to degradation. However, we found that a prolonged
Table 4. Unhydrolyzable organic carbon contents during incubation trials.
stabilization/humification process influenced its deSample
U†-TOC‡
U-TEC
U-HAC
U-NHC
U-HUC
gradability, as confirmed by the reduced presence of
g
the labile fraction with respect to the other samples.
SRM§
T0¶ 7.30 ± 0.55a# 4.35 ± 0.27b 3.12 ± 0.22b 1.52 ± 0.34b 2.95 ± 0.60a
Although this in itself is not a new finding because
T6
6.97 ± 0.75a 1.82 ± 0.37a 1.12 ± 0.3a
0.7 ± 0.47a 5.15 ± 0.82b
it
was previously shown that HAs were less degraded
SFC
T0
7.25 ± 0.05a
3.6 ± 0.36b 2.32 ± 0.57b 1.27 ± 0.58b 3.65 ± 0.36a
when
copious nutrients were present in the system
T6
6.77 ± 0.62a
1.4 ± 0.13a 0.95 ± 0.20a 0.45 ± 0.32a 5.37 ± 0.62b
(Filip
and
Kubát, 2001; Filip and Tesařová, 2004),
SEC
T0
7.57 ± 0.32b
2.7 ± 0.30b
2 ± 0.25b
0.7 ± 0.40a 4.87 ± 0.45a
the common belief has been that compost should be
T6
5.97 ± 0.50a 1.75 ± 0.30a
1 ± 0.22a
1 ± 0.37b 4.22 ± 0.30a
† Prefix U indicates unhydrolyzable fraction.
obtained after long composting periods because the
‡ FAC, fulvic acid carbon; HAC, humic acid carbon; HUC, humic carbon; NHC,
formation of evolved humified material takes a long
nonhumified carbon; TEC, total extractable carbon; TOC, total organic carbon.
time (Chen and Inbar, 1993). However, our results in§ Soil incubated with raw material (SRM), fresh compost (SFC), and evolved compost (SEC).
dicate that the contribution of compost to soil humus
¶ T0, T6: soil sampled after 0 and 6 mo of incubation.
depends on the compost U-HA content and on the
# Means followed in the same column by the same letter are not statistically different
presence of the labile fraction that is able to modulate
(p < 0.05) according to Tukey test.
1614
Journal of Environmental Quality • Volume 37 • July–August 2008
Fig. 4. Diffuse reflectance infrared fourier transformed spectra for humic acids (left) and unhydrolyzable-humic acid (right) extracted from soils
incubated with raw material (SRM), fresh compost (SFC), and evolved compost (SEC) after 6 mo of incubation.
the preservation of the recalcitrant fraction. Therefore, RM
and FC contribute to soil humus better than EC due to the
presence of the labile fraction. In addition, the labile fraction
plays an important role in soil structure formation and as nutrient (Swift, 1991).
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Journal of Environmental Quality • Volume 37 • July–August 2008